The DNA replication of two small Escherichia coli plasmids, Colicin E1 (Col E1) and P15A, and derivatives of the R plasmid R100.1 will be studied. In particular we will define the origin of replication of Col E1 with respect to primary and secondary structure. To this end we will determine the nucleotide sequence of the origin of replication of the plasmids P15A and mutants of Col E1. These nucleotide sequences will be compared with the known sequence of the Col El origin and the important structural features will be deduced. Mutants of both the host and Col El will be selected and extracts from mutant hosts will be used to purify the missing proteins by complementation of in vitro replication. The stage of replication at which these proteins operate will be determined using the in vitro replication system. The interaction of proteins involved in initiation of replication with the Col El origin and mutant Col El origins will be studied. These studies will allow the determination of the nucleotide sequence requirement of an origin of replication and provide insight into the control of DNA replication. These studies will be extended to include the R plasmid R100.1 A smaller derivative will be isolated by coupling the region containing the origin of replication and any genes required for replication to a selective marker. This smaller plasmid will be used to locate precisely the origin of replication and to select temperature-sensitive replication mutants. An in vitro replication system for this plasmid will be developed. Deletion mapping will be used to construct a fine structure map of the DNA replication genes. The function of these genes in the initiation of E. coli chromosome replication during integrative suppression of a E. coli dnaA mutant will be determined. The ultimate goal of these studies is to establish a system to study the control mechanisms of DNA replication. The effect of positive and/or negative control elements on the initiation of DNA replication will be studied in this system.